Modern systems biology and synthetic bioengineering face a grand challenge in relating the genetic components of a natural or engineered system to its integrated behavior; this is often referred to as the genotype to phenotype challenge. It involves the fundamental unsolved problem of relating the genotype-- which has a well-defined, generic, digital representation -- to the phenotype- which has a poorly-defined, ad hoc, analog representation. Without a rigorous generic definition of phenotype to provide the context for a deep understanding of the relation between genotype and phenotype, we are at a loss to know how many qualitatively distinct phenotypes are in an organism's repertoire or the relative fitness of the phenotypes in different environments. These are practical challenges for clinicians attempting to develop therapeutic strategies to treat pathology and bioengineers wishing to redirect normal cellular functions for biotechnological purposes. The projects of this proposal have been specifically selected to address this fundamental problem in two ways. First, they will serve as a test of the general applicability of a newly developed approach for relating genotype to phenotype that involves the first-of-its-kind generic characterization of a systems phenotypic repertoire. With this approach, phenotypes are identified and enumerated, their relative fitness compared, and their tolerance to phenotypic change measured. Although proof of concept has been established, the systems to which the method has been applied are few. Thus, applications to a very diverse selection of systems are needed to demonstrate the general utility of this innovative methodology. The specific aims of this proposal, which have been selected with this goal in mind, are to: (1) relate hemolytic anemia in diabetics with variants of the glucose-6-phosphate dehydrogenase enzyme to levels of oxidative stress, (2) uncover design principles for reversible pathways, (3) model a global transcription factor network implicated in growth-phase progression of bacteria, and (4) compare alternative gene circuitry for toxin-antitoxin and restriction-modification systems. Second, the characterization of their phenotypic repertoire will reveal underlying system design principles and improve basic understanding for general classes of gene circuitry and biochemical networks that are found in all organisms from bacteria to humans. The outline for the treatment in each case is as follows: Specific kinetic models are formulated based on known or suspected molecular elements and interactions; several criteria for functional effectiveness are identified that are relevant for the system in question; the complex nonlinear models are decomposed into a set of simpler submodels, each manifesting a qualitatively-distinct phenotype; the integrated behavior of each submodel is exhaustively analyzed and evaluated according to the criteria for functional effectiveness; the relative fitness of the phenotypes is established by quantitative comparison; results are interpreted in terms of effective design for specific functions; and, finally, physiological and pathological significance are addressed and predictions are made for experimental testing. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page